Functional inactivation of genes through the expression of synthetic genetic elements comprising all or a part of the gene to be inactivated is known in the art. At least four mechanisms exist by which expression of such specific genetic elements can result in inactivation of their corresponding gene. These are interference with protein function by polypeptides comprising nonfunctional or partly nonfunctional analogs of the protein to be inhibited or a portion thereof, interference with mRNA translation by complementary anti-sense RNA or DNA, destruction of mRNA by anti-sense RNA coupled with ribozymes, and interference with mRNA by RNA sequences homologous to a portion of the mRNA representing an important regulatory sequence.
Although gene suppression is quite useful for scientific studies of gene function and holds considerable promise for certain applications in disease therapy and genetic modification of plants and animals, current methods for identifying effective synthetic genetic elements (SGEs) are time consuming and arduous. Interference by dominant negative mutant proteins, for example, either requires extensive knowledge about the functional domain structure of the protein so that reasonably promising candidate mutant proteins can be prepared, or necessitates individual preparation and screening of numerous candidate mutant proteins. Antisense RNA and competitive homologous RNA similarly require extensive individual preparation and screening of candidate inhibitory sequences, absent considerable knowledge about important specific sequences within the RNA.
There is, therefore, a need for generalized methods for identifying and isolating SGEs that will allow simplified determination of effective elements without undue experimentation or extensive structure/function knowledge. An ideal method would allow simultaneous analysis of multiple possible candidate SGEs, regardless of their mechanism of action.
The present invention facilitates drug discovery by providing a method for identifying agents that selectively confer a desired phenotype on a target cell, comprising:
(i) transfecting subtractive cells with a library of expression vectors comprising a variegated population of coding sequences for potential synthetic genetic elements (SGEs);
(ii) isolating those SGE vectors of the SGE library that do confer the desired phenotype, or a phenotype that interferes with the detection thereof, to the subtractive cells;
(iii) transfecting target cells with the sub-population of SGE vectors isolated in step (ii); and
(iv) isolating those SGE vectors that confer the desired phenotype to the target cell.
One aspect of the subject method relates to a method for identifying agents with selective antiproliferative activity for a target cell, comprising:
(i) transfecting subtractive cells with a library of expression vectors comprising a variegated population of coding sequences for potential synthetic genetic elements (SGEs);
(ii) isolating those SGE vectors of the SGE library that are not lethal to the subtractive cells;
(iii) transfecting target cells with the non-lethal SGE vectors isolated in step (ii); and
(iv) isolating those SGE vectors that are lethal to the target cell.
In preferred embodiments, the method is carried out using target and subtractive cells that are eukaryotic cells, more preferably mammalian cells. In certain embodiments, one or both of the target and subtractive cells are human cells.
In certain embodiments, the target is a transformed cell, and the subtractive cell is an untransformed cell.
In other embodiments, the target is a cell infected with a virus, and the subtractive cell is an uninfected cell.
In preferred embodiments, the expression vectors are viral vectors, and more preferably retroviral vectors.
The SGE library can be generated from a normalized cDNA library, a subtractive cDNA library, or a combination thereof.
By random incorporation, or directional cloning, the library can be generated to include SGE having a sense oriented sequence encoding a peptide, and/or an antisense-oriented sequence encoding an antisense RNA.
Another aspect of the invention relates to formulations of the SGEs identified according to the subject method. For example, the present invention provides a synthetic oligonucleotide having a nucleotide sequence from about 12 nucleotides to all of the nucleotide sequence of an antisense RNA encoded by an SGE identified according to the subject method. In other embodiments, the invention provides an isolated peptide encoded by an SGE identified according to the subject method, or a peptidomimetic thereof.